The present invention relates to methods for using Chinese hamster ovary (CHO) cells for the anchorage-dependent and suspension-culture propagation of coronaviruses, including bovine coronavirus.
Bovine coronavirus (BCV) is a common cause of neonatal calf diarrhea, frequently afflicting calves aged two to four weeks. As a primary pathogen, BCV generally induces only a mild diarrhea. When combined with a secondary bacterial infection, however, BCV may become a major contributor to mortality in calves one month old and younger.
More recently, BCV has been implicated as a potential respiratory pathogen of older cattle, and it has been associated with outbreaks of winter dysentery in dairy cattle, as evidenced by the presence of BCV in the feces and BCV antibody seroconversion during such outbreaks.
There thus has been a need for culture systems to propagate BCV for the production of vaccines to prevent such cattle diseases.
Traditionally, BCV has been difficult to grow in cell culture, requiring an adaptation period following isolation. Many established cell lines are not suitable for growth of BCV, and much of the successfully reported propagation involves the use of primary cell cultures, which are unsuitable for large scale production. Furthermore, most coronaviruses show marked tissue tropism and will grow only in cells of the natural host species. See, e.g., Fleming et al., Adv. Exp. Med. Biol. 218:333-342 (1987); Sussman et al., Adv. Exp. Med. Biol. 218:399-410 (1987). Thus BCV is commonly grown in fetal bovine kidney cells, although it would be desirable to grow the virus instead in a more convenient established cell line.
In view of the economic importance of preventing BCV infections in cattle and the difficulties inherent in use of the cell culture systems commonly used to propagate the virus for vaccine production, there is a need for improved methods for growing BCV in culture.
The present invention fills the foregoing need by providing methods for the propagation of bovine coronavirus (BCV) in Chinese hamster ovary (CHO) cells. More particularly, this invention provides a method for propagating BCV comprising culturing CHO cells infected with BCV under conditions in which multiplication of the virus occurs.
In one embodiment, the CHO cells are CHO-K1 cells. In another, the BCV propagated is American Type Culture Collection strain ATCC VR-874. In another embodiment the CHO cells are grown in anchorage-dependent culture, while in another embodiment they are grown in suspension culture.
All references cited herein are hereby incorporated herein in their entirety by reference.
The present invention is based upon the surprising discovery that among various cell lines examined, CHO cells were uniquely suitable for efficiently growing BCV. Such cells were found to be particularly suitable for growing the virus in suspension culture, although they could also be used effectively attached to a suitable substrate.
Growth of BCV in CHO cells attached to a substrate can be carried out in all of the standard containers, including but not limited to tissue culture plates and flasks, roller bottles, and capillary assemblies or packed bed bioreactors in which BCV-infected cells attached to either capillary tubes or to other appropriate support matrices, e.g., glass beads or polymeric foams, are perfused with an appropriate culture medium, thereby permitting continuous harvesting of the virus from medium emerging from the capillary assembly or bioreactor. In the bioreactor systems, BCV-infected substrate-dependent CHO cells can be attached to polymeric microparticles. Alternatively, the CHO cells can be adapted by standard methods to grow independent of any substrate, maintained in suspension by agitation.
Culture medium that can be used in the present invention includes any of the media well known in the art to be useful for culturing CHO cells, including, e.g., Dulbecco""s modified Eagle""s medium (DMEM) and Glasgow""s modified Eagle""s medium (GMEM). Typically, such media are fortified by addition of an animal serum as a source of additional nutrients and growth factors. Examples of such animal sera suitable for use in this invention include, e.g., fetal calf serum (FCS) and adult bovine serum (ABS).
The present invention may also be practiced, however, using defined media, wherein the CHO cells are entirely weaned from the use of any animal serum. One example of such a defined medium that can be used is a basal medium such as DMEM containing hormonal and non-serum supplements, including albumin, insulin, transferrin and tryptose. Cells can be adapted to such a defined medium by continual passage in standard medium containing gradually reduced levels of serum, followed by transfer to mixtures of low serum and defined medium containing an increasing proportion of the defined medium.
The methods of this invention are applicable to the propagation of any BCV strain. Although a BCV called the Mebus strain and a field isolate from the United Kingdom were used to illustrate the invention in the Examples below, other isolates or other known strains could be used as well. Such known strains include, e.g., strains PQ, DB2, DBA, SD, 216XF, CN, BE, AW, OHC, SDC, JAZ, TS, BM, BW,L9, G110, F15, S1, S2, and CK.
Any CHO cell line can be used, including but not limited to the A2, A2H, XrS6, CHO-K1, CHO/dhFr, RR-CHOK1, UT-1, P22, CHO-1C6, Lec1, Lec2, Lec8, Pro-5, and DUKXB1 lines, although the CHO-K1 line deposited with the American Type Culture Collection under Accession No. ATCC CCL 61 is preferred. Similarly, a wide range of BCV can be propagated in the CHO cells, although propagation of the Mebus strain of BCV originally isolated from the feces of a calf afflicted with diarrhea in Nebraska, U.S.A. is described below for purposes of illustration of this invention.
To maximize the yield of virus produced, the CHO cells are preferably adapted to suspension culture using standard techniques, one example of which is illustrated below.